Prefoldin 4 in the treatment and diagnosis of cancer

ABSTRACT

This invention provides methods employing prefoldin-4 (PFDN-4) nucleic acid and polypeptide sequences to detect cancer or a propensity to develop cancer, to monitor the efficacy of a cancer treatment, and/or for prognostic applications. Further, the invention provides methods of identifying inhibitors of PfDN-4 and methods of treating cancer by inhibiting the expression and/or activity of PFDN-4.

CROSS-REFERENCE TO RELATED APPLICATIONS

This applications claims benefit of U.S. Provisional Application No.60/692,847, filed Jun. 20, 2005, which application is hereinincorporated by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made with Government support under grant No. CA45234awarded by the National Institutes of Health. The Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

Prefoldin 4 (PFDN-4) is a 134 amino acid protein having ahelix-loop-helix domain. The protein is a subunit of the heterohexamericchaperone protein prefoldin, a cytoplasmic protein that assists in thecorrect folding of other proteins, such as actin and tubulin, which aremajor components of the cellular cytoskeleton. Human PFDN-4 has beenlocalized to chromosome region 20q13. This region is frequentlyamplified in a variety of human cancers, including breast, ovarian, andcervical cancer. The sequence of the breast cancer amplicon has beenanalyzed (see, e.g., Collins et al., Genome Res. 11:1034-1042, 2001).Although PFDN-4 was identified as one of the genes present in theamplified region and was one of the genes shown to be overexpressed incell lines in which the amplicon was present, there was no evidence thatPFDN-4 played a role in tumorigenicity.

BRIEF SUMMARY OF THE INVENTION

The current invention is based on the discovery that PFDN-4 is amplifiedduring tumor progression and that overexpression of the proteinincreases tumor growth. PFDN-4 nucleic acid and protein sequences areamplified and over-expressed in cancers, e.g. pancreatic neuroendocrinetumors, breast cancer, ovarian cancer, cervical cancer, gastricadenocarcinomas, uroepithelial tumors, pancreatic islet tumors and othercancers, including those that have copy number gains in 20q13.Accordingly, the invention provides methods of identifying inhibitors ofPFDN-4 and methods of treating cancer, e.g., by inhibiting theexpression and/or activity of PFDN-4. The invention also providesmethods to detect cancer or a propensity to develop cancer, to monitorthe efficacy of a cancer treatment, and/or of using the sequence forprognostic applications.

Thus, in one aspect, the invention provides a method of identifying amodulator of expression of a cancer-associated polypeptide, the methodcomprising the steps of: (i) contacting a cell that expresses PFDN-4,e.g., SEQ ID NO:2, with a candidate modulator; and (ii) determining thelevel of expression of PFDN-4. In some embodiments, the modulator is anucleic acid such as a ribozyme, antisense nucleic acid, or an siRNA

The invention also provides a method for identifying a compound thatmodulates a cancer-associated polypeptide, the method comprising thesteps of: (i) contacting the compound with a PFDN-4 polypeptide, (e.g.,SEQ ID NO:2); and (ii) determining the functional effect of the compoundupon the polypeptide. The compounds can be, e.g., a small organicmolecule or an antibody.

In another aspect, the invention provides a method of inhibitingproliferation of a cancer cell that overexpresses a PFDN-4 polypeptide,e.g., a PFDN-4 having the amino acid sequence of SEQ ID NO:2, the methodcomprising the step of contacting the cancer cell with a therapeuticallyeffective amount of an inhibitor of the PFDN-4 polypeptide. In someembodiments, the cell has an amplification of 20q13. An inhibitor canbe, e.g., a small organic molecule, an antibody, an antisense molecule,a ribozyme, or an siRNA molecule.

In another aspect, the invention provides a method of detecting cancerin a biological sample from a patient, the method comprising: contactingthe sample with a polynucleotide that selectively hybridizes to anucleic acid sequence encoding a PFDN-4 polypeptide, e.g., a PFDN-4polypeptide having the amino acid sequence of SEQ ID NO:2; anddetermining an increase in the level of the nucleic acid sequence,relative to normal, thereby detecting the presence of cancer in thepatient. In some embodiments, the cancer comprises cells that have anamplification in the chromosomal region 20q13. The method can be used todetect many different cancers, including ovarian cancer, breast cancer,cervical cancer, gastric adenocarcinomas, uroepithelial tumors, andislet tumors.

In some embodiments, the detecting step comprises detecting an increasein copy number of the gene that encodes the PFDN-4 polypeptide. In otherembodiments, the detecting step comprises detecting an mRNA that encodesPFDN-4.

In some applications of the methods, the patient is suspected of havingcancer. In other applications, the patient is undergoing a therapeuticregimen to treat cancer.

The invention also provides a method of detecting cancer in a biologicalsample from a patient, the method comprising: detecting an increase inthe level of a PFDN-4 polypeptide, e.g., a PFDN-4 polypeptide having theamino acid sequence of SEQ ID NO:2, relative to normal, therebydetecting the presence of cancer in the patient. Typically, the level ofthe polypeptide is determining using an immunoassay. In otherembodiments, PFDN-4 activity is used to determine whether an increase inPFDN-4 levels is present.

The invention also provides a method of monitoring the efficacy of atherapeutic treatment of cancer, the method comprising the steps of: (i)providing a biological sample from a patient undergoing the therapeutictreatment; and (ii) detecting the level of: a PFDN-4 polypeptide, e.g.,a PFDN-4 polypeptide having an amino acid sequence of SEQ ID NO:2, or ofa nucleic acid that encodes the polypeptide, in the biological samplecompared to a level in a biological sample from the patient prior to, orearlier in, the therapeutic treatment, thereby monitoring the efficacyof the therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides exemplary data showing prefoldin subunit expressionlevels in different βTC cell lines A, PFDN-4 characterization βTC celllines derived from primary tumors (1B, 3, 4, 6, and 20) and UniversalMouse Reference RNA as positive control. B, Expression levels of sixPrefoldin component genes by qRT-PCR in βTC3, a cee line lackingamplification in mChr_(—)2.

FIG. 2 provides exemplary data showing characterization of βTC cellclones overexpressing PFDN-4 (Low, P4c3; high, P4c4) compared tocontrols (empty vector, C1; cells overexpressing a linked gene, Dok4).Top, western blot for PFDN-4 levels is shown. Percentages ofproliferating cells were determined by BrdU incorporation whileapoptosis rates were determined by cleaved caspase-3 immunodetection. *,p<0.001.

FIG. 3 provides exemplary data showing tumor growth of two βTC celllines overexpressing PFDN-4 by 10× and 50× compared with empty vectorcell line. Tumor size was measured with a caliper and the volume of aspheroid was calculated in cubic millimeters.

FIG. 4 provides exemplary data showing the correlation between PFDN-4expression levels and DNA copy number in 19 human breast cancer celllines. PFDN-4 expression levels were determined by qRT-PCR and DNA copynumber in 20q13.2 genomic region by array Comparative GenomeHybridization (aCGH). Each spot represents a different cell line. ThePearsons correlation coefficient between expression and copy number is0.85 (where 0.3 is the significance threshold).

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The present invention provides methods, reagents, and kits fordiagnosing cancer, for prognostic uses, and for treating cancer. Theinvention is based upon the discovery that PFDN-4 polynucleotide andpolypeptides are amplified and/or overexpressed in cancer cells and playa role in tumor growth. Cancers that can be detected using PFDN-4reagents and/or treated by inhibiting PFDN-4 expression include breastcancer, ovarian cancer, cervical cancer, gastric adenocarcinomas,uroepithelial tumors, uterus adenoma, mesothelioma, follicular lymphoma,lung adenocarcinoma, lung squamous carcinoma, renal carcinoma,pancreatic adenoma, medulloblastoma, small cell lung cancer, acutelymphocytic leukemia (B-cell), acute lymphocytic leukemia (T-cell), andacute myelomonocytic leukemia.

PFDN-4 plays a role in protein folding as part of the prefoldin complex.Prefoldin polynucleotide and polypeptides sequences are known. Forexample, human PFDN-4 polynucleotide sequences are available under thereference sequences NM_(—)002623.2, BC010953.1, and U41816.1. Anexemplary human polypeptide sequence is available under the accessionnumber Q9NQP4. The OMIM reference number for human PFDN-4 is 604898 andthe UniGene number is Hs.91161.

The ability to detect cancer cells by virtue of detecting an increasedlevel of a PFDN-4 nucleic acid or polypeptide sequence is useful for anyof a large number of applications. For example, an increased level ofpolynucleotides or proteins in cells of patient can be used, alone or incombination with other diagnostic methods, to diagnose cancer in thepatient or to determine the propensity of a patient to develop cancer.The detection of PFDN-4 sequences can also be used to monitor theefficacy of a cancer treatment. For example, the level of a PFDN-4polypeptide or polynucleotide after an anti-cancer treatment is comparedto the level before the treatment. A decrease in the level of the PFDN-4polypeptide or polynucleotide after the treatment indicates efficacioustreatment.

An increased level or diagnostic presence of PFDN-4 can also be used toinfluence the choice of anti-cancer treatment, where, for example, theincreased level of PFDN-4 directly correlates with the aggressiveness ofthe cancer and accordingly, the selection of anti-cancer therapy.

In addition, the ability to detect cancer cells can be useful to monitorthe number or location of cancer cells in a patient, in vivo or invitro, for example, to monitor the progression of the cancer over time.In addition, the level of PFDN-4 can be statistically correlated withthe efficacy of particular anti-cancer therapies or with observedprognostic outcomes, thereby allowing the development of databases basedon which a statistically-based prognosis, or a selection of the mostefficacious treatment, can be made in view of a particular level ordiagnostic presence of PFDN-4.

The present invention also provides methods of identifying inhibitors ofPFDN-4 and methods for treating cancer by inhibiting PFDN-4 expressionor activity. In certain embodiments, the proliferation is inhibited in acancer cell that has an increase in copy number of PFDN-4 andoverexpresses PFDN-4. The proliferation is decreased by, for example,contacting the cell with an inhibitor of PFDN-4 transcription ortranslation, or an inhibitor of the activity of PFDN-4. Such inhibitorsinclude, but are not limited to, small molecule inhibitors, siRNAs,antisense polynucleotides, ribozymes, and dominant negative PFDN-4polynucleotides or polypeptides, and antibodies.

Definitions

The term “PFDN-4” refers to nucleic acid and polypeptide polymorphicvariants, alleles, mutants, and interspecies homologues that: (1) havean amino acid sequence that has greater than about 60% amino acidsequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequenceidentity to PFDN-4 sequence of SEQ ID NO:2 or over a region of at leastabout 20, 50, 75, 100, or 125 or more amino acids of SEQ ID NO:2; (2)bind to antibodies, e.g., polyclonal antibodies, raised against animmunogen comprising an amino acid sequence of SEQ ID NO:2, orconservatively modified variants thereof; (3) specifically hybridizeunder stringent hybridization conditions to a PFDN-4 nucleic acidsequence of SEQ ID NO: 1 or conservatively modified variants thereof; or(4) or have a nucleic acid sequence that has greater than about 90%,preferably greater than about 96%, 97%, 98%, 99%, or higher nucleotidesequence identity, preferably over a region of over a region of at leastabout 30, 50, 100, 200, or 300 or more nucleotides, to SEQ ID NO:1; or(5) have at least 25, often 50, 75, or 100, 110, 120, or more contiguousamino acid of SEQ ID NO:2; or at least 25, often 50, 75, 100, 150, 200,250, 300, or 350 or more contiguous nucleotides of SEQ ID NO:1. A PDN-4polynucleotide or polypeptide sequence is typically from a human, butmay be from other mammals, but not limited to, a non-human primate, arodent, e.g:, a mouse, rat, or hamster; a cow, a pig, a horse, a sheep,or other mammal. A “PFDN-4” polypeptide and a “PFDN-4” polynucleotideinclude both naturally occurring or recombinant forms.

A “full length” PFDN-4 protein or nucleic acid refers to a PFDN-4polypeptide or polynucleotide sequence, or a variant thereof, thatcontains all of the elements normally contained in one or more naturallyoccurring, wild type PFDN-4 polynucleotide or polypeptide sequences. The“full length” may be prior to, or after, various stages ofpost-translation processing or splicing, including alternative splicing.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains nucleic acids or polypeptides, e.g., of a PFDN-4protein, polynucleotide or transcript. Such samples are typically fromhumans, but include tissues isolated from non-human primates, orrodents, e.g., mice, and rats. Biological samples may also includesections of tissues such as biopsy and autopsy samples, frozen sectionstaken for histologic purposes, blood, plasma, serum, sputum, stool,tears, mucus, hair, skin, etc. Biological samples also include explantsand primary and/or transformed cell cultures derived from patienttissues.

“Providing a biological sample” means to obtain a biological sample foruse in methods described in this invention. Most often, this will bedone by removing a sample of cells from a patient, but can also beaccomplished by using previously isolated cells (e.g., isolated byanother person, at another time, and/or for another purpose), or byperforming the methods of the invention in vivo. Archival tissues,having treatment or outcome history, will be particularly useful.

The “level of PFDN-4 mRNA” in a biological sample refers to the amountof mRNA transcribed from an PFDN-4 gene that is present in a cell or abiological sample. The mRNA generally encodes a functional PFDN-4protein, although mutations may be present that alter or eliminate thefunction of the encoded protein. A “level of PFDN-4 mRNA” need not bequantified, but can simply be detected, e.g., a subjective, visualdetection by a human, with or without comparison to a level from acontrol sample or a level expected of a control sample.

The “level of PFDN-4 protein or polypeptide” in a biological samplerefers to the amount of polypeptide translated from PFDN-4 mRNA that ispresent in a cell or biological sample. The polypeptide may or may nothave PFDN-4 protein activity. A “level of PFDN-4 protein” need not bequantified, but can simply be detected, e.g., a subjective, visualdetection by a human, with or without comparison to a level from acontrol sample or a level expected of a control sample.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specifiedregion, when compared and aligned for maximum correspondence over acomparison window or designated region) as measured using a BLAST orBLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like).Such sequences are then said to be “substantially identical.” Thisdefinition also refers to, or may be applied to, the compliment of atest sequence. The definition also includes sequences that havedeletions and/or additions, as well as those that have substitutions, aswell as naturally occurring, e.g., polymorphic or allelic variants, andman-made variants. As described below, the preferred algorithms canaccount for gaps and the like. Preferably, identity exists over a regionthat is at least about 25 amino acids or nucleotides in length, or morepreferably over a region that is 50-100 amino acids or nucleotides inlength.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof one of the number of contiguous positions selected from the groupconsisting typically of from 20 to 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Current Protocols in Molecular Biology (Ausubel et al., eds. 1995supplement)).

Preferred examples of algorithms that are suitable for determiningpercent sequence identity and sequence similarity include the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990). BLAST and BLAST 2.0 are used, with the parameters describedherein, to determine percent sequence identity for the nucleic acids andproteins of the invention. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/). This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, e.g.,for nucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid (protein) sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915)). For the purposes of this invention, theBLAST2.0 algorithm is used with the default parameters and the filteroff.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001. Log valuesmay be large negative numbers, e.g., 5, 10, 20, 30, 40, 40, 70, 90, 110,150, 170, etc.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, e.g., where the two peptides differonly by conservative substitutions. Another indication that two nucleicacid sequences are substantially identical is that the two molecules ortheir complements hybridize to each other under stringent conditions, asdescribed below. Yet another indication that two nucleic acid sequencesare substantially identical is that the same primers can be used toamplify the sequences.

A “host cell” is a naturally occurring cell or a transformed cell thatcontains an expression vector and supports the replication or expressionof the expression vector. Host cells may be cultured cells, explants,cells in vivo, and the like. Host cells may be prokaryotic cells such asE. coli, or eukaryotic cells such as yeast, insect, amphibian, ormammalian cells such as CHO, HeLa, and the like (see, e.g., the AmericanType Culture Collection catalog or web site, www.atcc.org).

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein or nucleic acid that is thepredominant species present in a preparation is substantially purified.In particular, an isolated nucleic acid is separated from some openreading frames that naturally flank the gene and encode proteins otherthan protein encoded by the gene. The term “purified” in someembodiments denotes that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. Preferably, it meansthat the nucleic acid or protein is at least 85% pure, more preferablyat least 95% pure, and most preferably at least 99% pure. “Purify” or“purification” in other embodiments means removing at least onecontaminant from the composition to be purified. In this sense,purification does not require that the purified compound be homogenous,e.g., 100% pure.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers, those containing modified residues, and non-naturallyoccurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an a carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs may have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical or associated, e.g., naturallycontiguous, sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode mostproteins. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to another of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes silentvariations of the nucleic acid. One of skill will recognize that incertain contexts each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, often silent variations of a nucleicacid which encodes a polypeptide is implicit in a described sequencewith respect to the expression product, but not with respect to actualprobe sequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention typically conservativesubstitutions for one another: 1) Alanine (A), Glycine (G); 2) Asparticacid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7)Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see,e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3^(rd) ed., 1994) and Cantor & Schimmel,Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains. Domains are portions ofa polypeptide that often form a compact unit of the polypeptide and aretypically 25 to approximately 500 amino acids long. Typical domains aremade up of sections of lesser organization such as stretches of β-sheetand α-helices. “Tertiary structure” refers to the complete threedimensional structure of a polypeptide monomer. “Quaternary structure”refers to the three dimensional structure formed, usually by thenoncovalent association of independent tertiary units.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” or grammaticalequivalents used herein means at least two nucleotides covalently linkedtogether. Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10,12, 15, 25, 30, 40, 50 or more nucleotides in length, up to about 100nucleotides in length. Nucleic acids and polynucleotides are a polymersof any length, including longer lengths, e.g., 200, 300, 500, 1000,2000, 3000, 5000, 7000, 10,000, etc. A nucleic acid of the presentinvention will generally contain phosphodiester bonds, although in somecases, nucleic acid analogs are included that may have alternatebackbones, comprising, e.g., phosphoramidate, phosphorothioate,phosphorodithioate, or O-methylphophoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress); and peptide nucleic acid backbones and linkages. Other analognucleic acids include those with positive backbones; non-ionicbackbones, and non-ribose backbones, including those described in U.S.Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC SymposiumSeries 580, Carbohydrate Modifications in Antisense Research, Sanghui &Cook, eds.. Nucleic acids containing one or more carbocyclic sugars arealso included within one definition of nucleic acids. Modifications ofthe ribose-phosphate backbone may be done for a variety of reasons,e.g., to increase the stability and half-life of such molecules inphysiological environments or as probes on a biochip. Mixtures ofnaturally occurring nucleic acids and analogs can be made;alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.

A variety of references disclose such nucleic acid analogs, including,for example, phosphoramidate (Beaucage et al., Tetrahedron 49(10):1925(1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970);Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl.Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984),Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al.,Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al., NucleicAcids Res. 19:1437 (1991); and U.S. Pat. No. 5,644,048),phosphorodithioate (Briu et al., J. Am. Chem. Soc. 11 1:2321 (1989),O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press), and peptidenucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc.114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992);Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996),all of which are incorporated by reference). Other analog nucleic acidsinclude those with positive backbones (Denpcy et al., Proc. Natl. Acad.Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew.Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem.Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597(1994); Chapters 2 and 3, ASC Symposium Series 580, “CarbohydrateModifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook;Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffset al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743(1996)) and non-ribose backbones, including those described in U.S. Pat.Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S.Sanghui and P. Dan Cook. Nucleic acids containing one or morecarbocyclic sugars are also included within one definition of nucleicacids (see Jenkins et al., Chem. Soc. Rev. (1995) pp 169-176). Severalnucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997page 35. All of these references are hereby expressly incorporated byreference.

Other analogs include peptide nucleic acids (PNA) which are peptidenucleic acid analogs. These backbones are substantially non-ionic underneutral conditions, in contrast to the highly charged phosphodiesterbackbone of naturally occurring nucleic acids. This results in twoadvantages. First, the PNA backbone exhibits improved hybridizationkinetics. PNAs have larger changes in the melting temperature (T_(m))for mismatched versus perfectly matched basepairs. DNA and RNA typicallyexhibit a 2-4° C. drop in T_(m) for an internal mismatch. With thenon-ionic PNA backbone, the drop is closer to 7-9° C. Similarly, due totheir non-ionic nature, hybridization of the bases attached to thesebackbones is relatively insensitive to salt concentration. In addition,PNAs are not degraded by cellular enzymes, and thus can be more stable.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. As will be appreciated by those in the art, thedepiction of a single strand also defines the sequence of thecomplementary strand; thus the sequences described herein also providethe complement of the sequence. Unless otherwise indicated, a particularnucleic acid sequence also implicitly encompasses conservativelymodified variants thereof (e.g., degenerate codon substitutions) andcomplementary sequences, as well as the sequence explicitly indicated.The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid,where the nucleic acid may contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases, including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine,isoguanine, etc. “Transcript” typically refers to a naturally occurringRNA, e.g., a pre-mRNA, hnRNA, or mRNA. As used herein, the term“nucleoside” includes nucleotides and nucleoside and nucleotide analogs,and modified nucleosides such as amino modified nucleosides. Inaddition, “nucleoside” includes non-naturally occurring analogstructures. Thus, e.g. the individual units of a peptide nucleic acid,each containing a base, are referred to herein as a nucleoside.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins or otherentities which can be made detectable, e.g., by incorporating aradiolabel into the peptide or used to detect antibodies specificallyreactive with the peptide. The labels may be incorporated into thePFDN-4 nucleic acids, proteins and antibodies at any position. Anymethod known in the art for conjugating the antibody to the label may beemployed, e.g., using methods described in Hermanson, BioconjugateTechniques 1996, Academic Press, Inc., San Diego.

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe. Alternatively,method using high affinity interactions may achieve the same resultswhere one of a pair of binding partners binds to the other, e.g.,biotin, streptavidin.

As used herein a “nucleic acid probe or oligonucleotide” is defined as anucleic acid capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,C, or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in a probe may be joined by a linkage other than aphosphodiester bond, so long as it does not functionally interfere withhybridization. Thus, e.g., probes may be peptide nucleic acids in whichthe constituent bases are joined by peptide bonds rather thanphosphodiester linkages. It will be understood by one of skill in theart that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are preferably directly labeledas with isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.Diagnosis or prognosis may be based at the genomic level, or at thelevel of RNA or protein expression.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, e.g., recombinant cells express genes that are not foundwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under expressed or notexpressed at all. By the term “recombinant nucleic acid” herein is meantnucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid, e.g., using polymerases and endonucleases,in a form not normally found in nature. In this manner, operably linkageof different sequences is achieved. Thus an isolated nucleic acid, in alinear form, or an expression vector formed in vitro by ligating DNAmolecules that are not normally joined, are both considered recombinantfor the purposes of this invention. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, i.e., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. Similarly, a “recombinantprotein” is a protein made using recombinant techniques, i.e., throughthe expression of a recombinant nucleic acid as depicted above.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not normally found in the same relationship toeach other in nature. For instance, the nucleic acid is typicallyrecombinantly produced, having two or more sequences, e.g., fromunrelated genes arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source.Similarly, a heterologous protein will often refer to two or moresubsequences that are not found in the same relationship to each otherin nature (e.g., a fusion protein).

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal is at least two times background, preferably 10 timesbackground hybridization. Exemplary stringent hybridization conditionscan be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42°C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and0.1% SDS at 65° C. For PCR, a temperature of about 36° C. is typical forlow stringency amplification, although annealing temperatures may varybetween about 32° C. and 48° C. depending on primer length. For highstringency PCR amplification, a temperature of about 62° C. is typical,although high stringency annealing temperatures can range from about 50°C. to about 65° C., depending on the primer length and specificity.Typical cycle conditions for both high and low stringency amplificationsinclude a denaturation phase of 90° C.-95° C. for 30 sec-2 min., anannealing phase lasting 30 sec.-2 min., and an extension phase of about72° C. for 1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al. (1990) PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.N.Y.).

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous reference, e.g., andCurrent Protocols in Molecular Biology, ed. Ausubel, et al.

The phrase “functional effects” in the context of assays for testingcompounds that modulate activity of a PFDN-4 protein includes thedetermination of a parameter that is indirectly or directly under theinfluence of the PFDN-4 cancer protein or nucleic acid, e.g., afunctional, physical, or chemical effect, such as the ability todecrease tumorigenesis. It includes protein-protein interactionactivity; cell growth on soft agar; anchorage dependence; contactinhibition and density limitation of growth; cellular proliferation;cellular transformation; growth factor or serum dependence; tumorspecific marker levels; invasiveness into Matrigel; tumor growth andmetastasis in vivo, including measurement of tumor growth and tumor“take” in a model system; mRNA and protein expression in cells,including those undergoing metastasis, and other characteristics ofcancer cells. “Functional effects” include in vitro, in vivo, and exvivo activities.

By “determining the functional effect” is meant assaying for a compoundthat increases or decreases a parameter that is indirectly or directlyunder the influence of a PFDN-4 protein sequence, e.g., functional,enzymatic, physical and chemical effects. Such functional effects can bemeasured by any means known to those skilled in the art, e.g., changesin spectroscopic characteristics (e.g., fluorescence, absorbance,refractive index), hydrodynamic (e.g., shape), chromatographic, orsolubility properties for the protein, measuring inducible markers ortranscriptional activation of the PFDN-4 protein; measuring bindingactivity or binding assays, e.g. binding to antibodies or other ligands,and measuring cellular proliferation. Determination of the functionaleffect of a compound on tumorigenesis can be performed using exemplaryassays disclosed above. The functional effects can be evaluated by manymeans known to those skilled in the art, e.g., microscopy forquantitative or qualitative measures of alterations in morphologicalfeatures, measurement of changes in RNA or protein levels for PFDN-4sequences, measurement of RNA stability, identification of downstream orreporter gene expression (CAT, luciferase, β-gal, GFP and the like),e.g., via chemiluminescence, fluorescence, colorimetric reactions,antibody binding, inducible markers, and ligand binding assays.

“Inhibitors” or “modulators” of PFDN-4 polynucleotide and polypeptidesequences are used to refer to inhibitory molecules or compoundsidentified using in vitro and in vivo assays of PFDN-4 polynucleotideand polypeptide sequences. Inhibitors are compounds that, e.g., bind to,partially or totally block activity, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate the activity orexpression of PFDN-4 proteins, e.g., antagonists. Inhibitors includesiRNA or antisense RNA, genetically modified versions of PFDN-4proteins, e.g., versions with altered activity, as well as naturallyoccurring and synthetic PFDN antagonists, antibodies, small chemicalmolecules and the like. Such assays for inhibitors and activatorsinclude, e.g., expressing the PFDN-4 protein in vitro, in cells, or cellmembranes, applying putative modulator compounds, and then determiningthe functional effects on activity, as described above.

Samples or assays comprising PFDN-4 proteins that are treated with apotential inhibitor are compared to control samples without theinhibitor, to examine the extent of inhibition. Control samples(untreated with inhibitors) are assigned a relative protein activityvalue of 100%. Inhibition of a PFDN-4 polypeptide is achieved when theactivity value relative to the control is about 80%, preferably 50%,more preferably 25-0%.

The phrase “changes in cell growth” refers to any change in cell growthand proliferation characteristics in vitro or in vivo, such as formationof foci, anchorage independence, semi-solid or soft agar growth, changesin contact inhibition and density limitation of growth, loss of growthfactor or serum requirements, changes in cell morphology, gaining orlosing immortalization, gaining or losing tumor specific markers,ability to form or suppress tumors when injected into suitable animalhosts, and/or immortalization of the cell. See, e.g., Freshney, Cultureof Animal Cells a Manual of Basic Technique pp. 231-241 (3^(rd) ed.1994).

“Tumor cell” refers to precancerous, cancerous, and normal cells in atumor.

“Cancer cells,” “transformed” cells or “transformation” in tissueculture, refers to spontaneous or induced phenotypic changes that do notnecessarily involve the uptake of new genetic material. Althoughtransformation can arise from infection with a transforming virus andincorporation of new genomic DNA, or uptake of exogenous DNA, it canalso arise spontaneously or following exposure to a carcinogen, therebymutating an endogenous gene. Transformation is associated withphenotypic changes, such as immortalization of cells, aberrant growthcontrol, nonmorphological changes, and/or malignancy (see, Freshney,Culture of Animal Cells a Manual of Basic Technique (3^(rd) ed. 1994)).

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.Typically, the antigen-binding region of an antibody or its functionalequivalent will be most critical in specificity and affinity of binding.See Paul, Fundamental Immunology.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, e.g., pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H) 1 by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab)′₂ dimer intoan Fab′ monomer. The Fab′ monomer is essentially Fab with part of thehinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). Whilevarious antibody fragments are defined in terms of the digestion of anintact antibody, one of skill will appreciate that such fragments may besynthesized de novo either chemically or by using recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments either produced by the modification of wholeantibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990))

For preparation of antibodies, e.g., recombinant, monoclonal, orpolyclonal antibodies, many technique known in the art can be used (see,e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al.,Immunology Today 4:72 (1983); Cole et al., pp. 77-96 in MonoclonalAntibodies and Cancer Therapy (1985); Coligan, Current Protocols inImmunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual(1988); and Goding, Monoclonal Antibodies: Principles and Practice (2ded. 1986)). Techniques for the production of single chain antibodies(U.S. Pat. No. 4,946,778) can be adapted to produce antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies. Alternatively, phage display technology can be used toidentify antibodies and heteromeric Fab fragments that specifically bindto selected antigens (see, e.g., McCafferty et al., Nature 348:552-554(1990); Marks et al., Biotechnology 10:779-783 (1992)).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

Identification of PFDN-4 Sequences in a Sample from a Patient

In one aspect of the invention, the expression levels of PFDN-4 aredetermined in different patient samples for which diagnostic orprognostic information is desired. That is, normal tissue may bedistinguished from cancerous or metastatic cancerous tissue, e.g.,cancerous or metastatic ovarian or breast tissuet; or cancer tissue ormetastatic cancerous tissue can be compared with corresponding tissuesamples from other patients, e.g., surviving cancer patients.

General Recombinant DNA Methods

This invention relies on routine techniques in the field of recombinantgenetics for the preparation of PFDN-4 for use in the invention and formethods of detecting PFDN-4. Basic texts disclosing the general methodsof use in this invention include Sambrook & Russell, Molecular Cloning,A Laboratory Manual (3rd Ed, 2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994-1999). Methods that areused to produce PFDN-4 for use in the invention may also be employed toproduce other polypeptides, e.g., candidate modulators, for use in theinvention. For nucleic acids, sizes are given in either kilobases (kb)or base pairs (bp). For proteins, sizes are given in kilodaltons (kDa)or amino acid residue numbers.

Methods for the Isolation and Expression of PFDN-4 Nucleotide Sequences

In general, the nucleic acid sequences encoding PFDN-4 and relatednucleic acid sequence homologs are cloned from cDNA and genomic DNAlibraries by hybridization with a probe, or isolated using amplificationtechniques with oligonucleotide primers. For example, sequences aretypically isolated from mammalian nucleic acid (genomic or cDNA)libraries by hybridizing with a nucleic acid probe, the sequence ofwhich can be derived from SEQ ID NOS:1. Amplification techniques usingprimers can also be used to amplify and isolate nucleic acids from DNAor RNA (see, e.g., section “detection of polynucleotides”, below).Suitable primers for amplification of specific sequences can be designedusing principles well known in the art (see, e.g., Dieffenfach &Dveksler, PCR Primer: A Laboratory Manual (1995)). These primers can beused, e.g., to amplify either the full length sequence or a probe thatis then used to identify PFDN-4 polynucleotides.

Nucleic acids encoding PFDN-4 can also be isolated from expressionlibraries using antibodies as probes. Such polyclonal or monoclonalantibodies can be raised using the sequence of SEQ ID NOs:2.

Synthetic oligonucleotides can also be used to construct PFDN-4 genesfor use as probes or for expression of protein. This method is performedusing a series of overlapping oligonucleotides, usually 40-120 bp inlength, representing both the sense and nonsense strands of the gene.These DNA fragments are then annealed, ligated and cloned.Alternatively, amplification techniques can be used with precise primersto amplify a specific subsequence of the nucleic acid. The specificsubsequence is then ligated into an expression vector.

To obtain high level expression of a cloned gene or nucleic acid, suchas cDNAs encoding PFDN-4, one typically subdlones a PFDN-4 nucleic acidinto an expression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Additional elements that are typicallyincluded in expression vectors also include a replicon that functions inE. coli, a gene encoding antibiotic resistance to permit selection ofbacteria that harbor recombinant plasmids, and unique restriction sitesin nonessential regions of the plasmid to allow insertion of eukaryoticsequences.

Suitable bacterial and eukaryotic expression systems promoters are wellknown in the art and described, e.g., in Sambrook & Russell, supra,Ausubel et al, supra. Bacterial expression systems for expressing thePFDN-4 protein include e.g., E. coli, Bacillus sp., and Salmonella.Eukaryotic expression systems include those for expressing sequences inmamralian cells, yeast, and insect cells. In one embodiment, theeukaryotic expression vector is a viral vector, e.g., an adenoviralvector, an adeno-associated vector, or a retroviral vector. Kits forprokaryotic and eukaryotic expression systems are commerciallyavailable.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of PFDN-4protein, which are then purified using standard techniques (see, e.g.,Scopes, Protein Purification: Principles and Practice (1982); U.S. Pat.No. 4,673,641; Ausubel et al., supra; and Sambrook et al., supra Guideto Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher,ed., 1990)). Transformation of eukaryotic and prokaryotic cells areperformed according to standard techniques (see, e.g., Morrison, J.Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Detection of Polynucleotides

The invention provides methods for detecting PFDN-4 polynucleotide andpolypeptide sequences, e.g., for the diagnosis and prognosis of cancer.Typically, the level of a PFDN-4 polynucleotide or polypeptide will bedetected in a biological sample. As noted above, a “biological sample”refers to a cell or population of cells or a quantity of tissue or fluidfrom an animal. Most often, the sample has been removed from an animal,but the term “biological sample” can also refer to cells or tissueanalyzed in vivo, i.e., without removal from the animal. Typically, a“biological sample” will contain cells from the animal, but the term canalso refer to noncellular biological material, such as noncellularfractions of blood, saliva, or urine, that can be used to measure thecancer-associated polynucleotide or polypeptide levels. Numerous typesof biological samples can be used in the present invention, including,but not limited to, a tissue biopsy, a blood sample, a saliva sample, ora nipple discharge.

As used herein, a “tissue biopsy” refers to an amount of tissue removedfrom an animal for diagnostic analysis. In a patient with cancer, tissuemay be removed from a tumor, allowing the analysis of cells within thetumor. “Tissue biopsy” can refer to any type of biopsy, such as needlebiopsy, fine needle biopsy, surgical biopsy, etc.

Detection of Copy Number

In one embodiment, diagnostic and prognostic detection of PFDN-4 incancer is evaluated by determining the copy number of PFDN-4, i.e., thenumber of DNA sequences in a cell encoding PFDN-4. Methods of evaluatingthe copy number of a particular gene are well known to those of skill inthe art, and include, inter alia, hybridization and amplification basedassays.

Hybridization-based Assays

Any of a number of hybridization based assays can be used to detect thecopy number of PFDN-4 in the cells of a biological sample. One suchmethod is by Southern blot. In a Southern blot, genomic DNA is typicallyfragmented, separated electrophoretically, transferred to a membrane,and subsequently hybridized to a cancer-associatedpolynucleotide-specific probe. Comparison of the intensity of thehybridization signal from the probe for the target region with a signalfrom a control probe for a region of normal genomic DNA (e.g., anonamplified portion of the same or related cell, tissue, organ, etc.)provides an estimate of the relative copy number of thecancer-associated gene. Southern blot methodology is well known in theart and is described, e.g., in Ausubel et al., or Sambrook et al.,supra.

An alternative means for determining the copy number of PFDN-4 in asample is by in situ hybridization, e.g., fluorescence in situhybridization, or FISH. In situ hybridization assays are well known(e.g., Angerer (1987) Meth. Enzymol 152: 649). Generally, in situhybridization comprises the following major steps: (1) fixation oftissue or biological structure to be analyzed; (2) prehybridizationtreatment of the biological structure to increase accessibility oftarget DNA, and to reduce nonspecific binding; (3) hybridization of themixture of nucleic acids to the nucleic acid in the biological structureor tissue; (4) post-hybridization washes to remove nucleic acidfragments not bound in the hybridization and (5) detection of thehybridized nucleic acid fragments.

The probes used in such applications are typically labeled, e.g., withradioisotopes or fluorescent reporters. Preferred probes aresufficiently long, e.g., from about 50, 100, or 200 nucleotides to about1000 or more nucleotides, so as to specifically hybridize with thetarget nucleic acid(s) under stringent conditions.

In numerous embodiments, “comparative probe” methods, such ascomparative genomic hybridization (CGH), are used to detect PFDN-4 geneamplification. In comparative genomic hybridization methods, a “test”collection of nucleic acids is labeled with a first label, while asecond collection (e.g., from a healthy cell or tissue) is labeled witha second label. The ratio of hybridization of the nucleic acids isdetermined by the ratio of the first and second labels binding to eachfiber in an array. Differences in the ratio of the signals from the twolabels, e.g., due to gene amplification in the test collection, isdetected and the ratio provides a measure of the PFDN-4 gene copynumber.

Hybridization protocols suitable for use with the methods of theinvention are described, e.g., in Albertson (1984) EMBO J. 3: 1227-1234;Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No.430,402; Methods in Molecular Biology, Vol. 33: In Situ HybridizationProtocols, Choo, ed., Humana Press, Totowa, N.J. (1994), etc.

Amplification-based Assays

In another embodiment, amplification-based assays are used to measurethe copy number of PFDN-4. In such an assay, the PFDN-4 nucleic acidsequences act as a template in an amplification reaction (e.g.,Polymerase Chain Reaction, or PCR). In a quantitative amplification, theamount of amplification product will be proportional to the amount oftemplate in the original sample. Comparison to appropriate controlsprovides a measure of the copy number of the cancer-associated gene.Methods of quantitative amplification are well known to those of skillin the art. Detailed protocols for quantitative PCR are provided, e.g.,in Innis et al. (1990) PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc. N.Y.). The known nucleic acidsequences for PFDN-4 (see, e.g., SEQ ID NO:1) is sufficient to enableone of skill to routinely select primers to amplify any portion of thegene. Suitable primers for amplification of specific sequences can bedesigned using principles well known in the art (see, e.g., Dieffenfach& Dveksler, PCR Primer: A Laboratory Manual (1995))

In preferred embodiments, a TaqMang based assay is used to quantify thecancer-associated polynucleotides. TaqMan® based assays use afluorogenic oligonucleotide probe that contains a 5′ fluorescent dye anda 3′ quenching agent. The probe hybridizes to a PCR product, but cannotitself be extended due to a blocking agent at the 3′ end. When the PCRproduct is amplified in subsequent cycles, the 5′ nuclease activity ofthe polymerase, e.g., AmpliTaq®, results in the cleavage of the TaqMan®probe. This cleavage separates the 5′ fluorescent dye and the 3′quenching agent, thereby resulting in an increase in fluorescence as afunction of amplification (see, for example, literature provided byPerkin-Elmer, e.g., www2.perkin-elmer.com).

Other suitable amplification methods include, but are not limited to,ligase chain reaction (LCR) (see, Wu and Wallace (1989) Genomics 4: 560,Landegren et al. (1988) Science 241: 1077, and Barringer et al. (1990)Gene 89: 117), transcription amplification (Kwoh et al. (1989) Proc.Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication(Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR,and linker adapter PCR, etc.

Detection of mRNA Expression

Direct Hybridization-based Assays

Methods of detecting and/or quantifying the level of PFDN-4 genetranscripts (mRNA or cDNA made therefrom) using nucleic acidhybridization techniques are known to those of skill in the art. Forexample, gexpression levels of PFDN-4 can also be analyzed by techniquesknown in the art, e.g., dot blotting, in situ hybridization, RNaseprotection, probing DNA microchip arrays, and the like. In oneembodiment, high density oligonucleotide analysis technology (e.g.,GeneChip™) is used to identify PFDN-4 sequences.

Amplification-based Assays

In another embodiment, a PFDN-4 transcript is detected usingamplification-based methods (e.g., RT-PCR). RT-PCR methods are wellknown to those of skill (see, e.g., Ausubel et al., supra). Preferably,quantitative RT-PCR, e.g., a TaqMan® assay, is used, thereby allowingthe comparison of the level of mRNA in a sample with a control sample orvalue.

Detection of PFDN-4 Polypeptide Sequences

Altered PFDN-4 expression or activity can also be detected by detectinglevels of PFDN-4 protein or activity. For example, detection of PfDN-4protein activity or expression can be used for diagnostic purposes or inscreening assays. In some embodiments, PFDN-4 level is convenientlydetermined using immunological assays to detect the level of PFDN-4polypeptides. The following section discusses immunological detection ofPFDN-4. The section also relates to generation and engineering oftherapeutic antibodies.

Immunological Detection PFDN-4

Antibodies can also be used to detect PFDN-4 or can be assessed in themethods of the invention for the ability to inhibit PFDN-4. PFDN-4 or afragment thereof may be used to produce antibodies specifically reactivewith PFDN-4. For example, a recombinant PFDN-4 or an antigenic fragmentthereof, is isolated as described herein. Recombinant protein is thepreferred immunogen for the production of monoclonal or polyclonalantibodies. Alternatively, a synthetic peptide derived from thesequences disclosed herein and conjugated to a carrier protein can beused as an immunogen. Naturally occurring protein may also be usedeither in pure or impure form. The product is then used to generateantibodies.

A general overview of the applicable technology can be found in Harlow &Lane, Antibodies: A Laboratory Manual (1988) and Harlow & Lane, UsingAntibodies (1999). Methods of producing polyclonal and monoclonalantibodies that react specifically with PFDN-4 are known to those ofskill in the art (see, e.g., Coligan, Current Protocols in Immunology(1991); Harlow & Lane, supra; Goding, Monoclonal Antibodies: Principlesand Practice (2d ed. 1986); and Kohler & Milstein, Nature 256:495-497(1975). Such techniques include antibody preparation by selection ofantibodies from libraries of recombinant antibodies in phage or similarvectors, as well as preparation of polyclonal and monoclonal antibodiesby immunizing rabbits or mice (see, e.g., Huse et al., Science246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989)). Suchantibodies can be used for diagnostic or prognostic applications, e.g.,in the detection of cancer, e.g., ovarian, breast cancer, cervicalcancer, pancreatic islet cancer, or for other cancers that exhibitincreased expression or activity of PFDN-4.

Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-PFDN-4proteins or even other related proteins from other organisms, using acompetitive binding immunoassay. Specific polyclonal antisera andmonoclonal antibodies will usually bind with a K_(d) of at least about0.1 mM, more usually at least about 1 μM, optionally at least about 0.1μM or better, and optionally 0.01 μM or better.

In some embodiments, a PFDN-4 antibody may be used for therapeuticapplications. For example, such an antibody may be conjugated to aprotein that facilitates entry into the cell. In one case, the antibodyenters the cell by endocytosis. In another embodiment, a nucleic acidencoding the antibody is administered to the individual or cell.

In one embodiment, the antibodies to the PFDN-4 protein are capable ofreducing or eliminating a biological function of PFDN-4 as is describedbelow. That is, the addition of anti-PFDN-4 antibodies (eitherpolyclonal or preferably monoclonal) to cancer tissue (or a cellpopulation containing cancererous cells) may reduce or eliminate thecancer. Generally, at least a 25% decrease in activity, growth, size orthe like is preferred, with at least about 50% being particularlypreferred and about a 95-100% decrease being especially preferred.

Often, the antibodies to the PFDN-4 proteins for therapeuticapplications are humanized antibodies (e.g., Xenerex Biosciences,Mederex, Inc., Abgenix, Inc., Protein Design Labs,Inc.) Humanized formsof non-human (e.g., murine) antibodies are chimeric molecules ofimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, a humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe framework (FR) regions are those of a human immunoglobulin consensussequence. The humanized antibody optimally also will comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992)). Humanization can be essentially performed followingthe method of Winter and co-workers (Jones et al., Nature 321:522-525(1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody.Accordingly, such humanized antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries (Hoogenboom & Winter, J. Mol.Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)). Thetechniques of Cole et al. and Boerner et al. are also available for thepreparation of human monoclonal antibodies (Cole et al., MonoclonalAntibodies and Cancer Therapy, p. 77 (1985) and Boerner et al., J.Immunol. 147(1):86-95 (1991)). Similarly, human antibodies can be madeby introducing of human immunoglobulin loci into transgenic animals,e.g., mice in which the endogenous immunoglobulin genes have beenpartially or completely inactivated. Upon challenge, human antibodyproduction is observed, which closely resembles that seen in humans inall respects, including gene rearrangement, assembly, and antibodyrepertoire. This approach is described, e.g., in U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and inthe following scientific publications: Marks et al., Bio/Technology10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison,Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); Lonberg& Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

Once PFDN-4-specific antibodies are available, binding interactions withPFDN-4 can be detected by a variety of immunoassay methods. PFDN-4 canbe detected and/or quantified using any of a number of well recognizedimmunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241;4,376,110; 4,517,288; and 4,837,168). For areview of the generalimmunoassays, see also Methods in Cell Biology: Antibodies in CellBiology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology(Stites & Terr, eds., 7th ed. 1991). Immunological binding assays (orimmunoassays) typically use an antibody that specifically binds to aprotein or antigen of choice (in this case PFDN-4 or antigenicsubsequence thereof). The immunoassays can be performed in any ofseveral configurations, which are reviewed extensively in EnzymeImmunoassay (Maggio, ed., 1980); and Harlow & Lane, supra.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled PFDN-4 polypeptide ora labeled anti-PFDN-4 antibody. Alternatively, the labeling agent may bea third moiety, such as a secondary antibody, that specifically binds tothe antibody/ antigen complex (a secondary antibody is typicallyspecific to antibodies of the species from which the first antibody isderived). Other proteins capable of specifically binding immunoglobulinconstant regions, such as protein A or protein G may also be used as thelabeling agent. These proteins exhibit a strong non-immunogenicreactivity with immunoglobulin constant regions from a variety ofspecies (see, e.g., Kronval et al., J. Immunol. 111:1401-1406 (1973);Akerstrom et al., J. Immunol. 135:2589-2542 (1985)). The labeling agentcan be modified with a detectable moiety, such as biotin, to whichanother molecule can specifically bind, such as streptavidin. A varietyof detectable moieties are well known to those skilled in the art.

Commonly used assays include noncompetitive assays, e.g., sandwichassays, and competitive assays. In competitive assays, the amount ofPFDN-4 present in the sample is measured indirectly by measuring theamount of a known, added (exogenous) PFDN-4 displaced (competed away)from an anti-PFDN-4 antibody by the unknown PFDN-4 present in a sample.Commonly used assay formats include immunoblots, which are used todetect and quantify the presence of protein in a sample. Other assayformats include liposome immunoassays (LIA), which use liposomesdesigned to bind specific molecules (e.g., antibodies) and releaseencapsulated reagents or markers. The released chemicals are thendetected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34-41 (1986)).

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent compounds (e.g., fluorescein isothiocyanate, Texas red,rhodamine, fluorescein, and the like), radiolabels, enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in anELISA), streptavidin/biotin, and calorimetric labels such as colloidalgold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.). Chemiluminescent compounds may also beused. For a review of various labeling or signal producing systems thatmay be used, see U.S. Pat. No. 4,391,904.

Detection of Activity

As appreciated by one of skill in the art, PFDN-4 activity can bedetected to evaluate expression levels or for identifying modulators ofactivity. The activity can be assessed using a variety of in vitro andin vivo assays to determine functional, chemical, and physical effects,e.g., measuring protein-protein interactions, measuring apoptosis,measuring transcription levels, measuring indicators of transformation,e.g., growth in soft agar, change in cell phenotype, ability to modifytumorigenesis, and the like. For example, PFDN-4 is a member of theprefoldin complex. Thus, in some embodiments, PFDN-4 activity can beassessed by monitoring prefoldin assembly and/or monitoring the abilityof PFDN-4 to interact with other components of prefoldin.

In other embodiments, as noted above, the ability of a test compound tomodulate PFDN-4 is tested by examining markers of cellulartransformation or apoptosis. In these embodiments, a candidate inhibitoris examined for the ability to inhibit transformation-associatedphenotypic changes of cells and/or apoptosis. Such tests may beperformed in vitro or in vivo. For example, a candidate compound may betested for the ability to counteract PFDN-4-associated decreases inapoptosis. Alternatively, a candidate compound may be tested for theability to decreased the enhanced tumor “take” rates associated withPFDN-4 overexpression using an animal model, such as a mouse model oftumorigenesis (see, e.g., the Examples section).

The PFDN-4 for the assay is often selected from a polypeptide having asequence of SEQ ID NO:2, or conservatively modified variants thereof.Alternatively, the PFDN-4 will be derived from a eukaryote and includean amino acid subsequence having amino acid sequence identity to SEQ IDNO:2. Generally, the amino acid sequence identity will be at least 70%,optionally at least 80%, or 90-95%. The PFDN-4 typically comprises atleast 10 contiguous amino acids, often at least 20, 50, or 100contiguous amino acids of SEQ ID NO:2. Optionally, the polypeptide ofthe assays will comprise or consist of a domain of PFDN-4, such as asubunit association domain, active site, and the like. Either a PFDN-4or a domain thereof can be covalently linked to a heterologous proteinto create a chimeric protein used in the assays described herein.

Activity assays of the invention are used to identify modulators thatcan be used as therapeutic agents, e.g., antibodies to PFDN-4 andantagonists of PFDN-4 activity Modulators of PFDN-4 activity are testedusing PFDN-4 polypeptides as described above, either recombinant ornaturally occurring. The protein can be isolated, expressed in a cell,expressed in tissue or in an animal, either recombinant or naturallyoccurring. For example, transformed cells can be used. Modulation istested using one of the in vitro or in vivo assays described herein.Activity can also be examined in vitro with soluble or solid statereactions, using a PFDN-4 fragment that binds to another protein, e.g,another component of the prefoldin complex.

In another embodiment, mRNA and/or protein expression levels can bemeasured to assess the effects of a test compound on PFDN-4. A host cellexpressing PFDN-4 is contacted with a test compound for a sufficienttime to effect any interactions, and then the level of mRNA or proteinis measured. The amount of time to effect such interactions may beempirically determined, such as by running a time course and measuringthe level of expression as a function of time. The amount of expressionmay be measured by using any method known to those of skill in the artto be suitable. For example, mRNA expression may be detected usingnorthern blots or polypeptide levels may be identified usingimmunoassays. Alternatively, transcription based assays using reportergenes may be used as described in U.S. Pat. No. 5,436,128, hereinincorporated by reference. The reporter genes can be, e.g.,chloramphenicol acetyltransferase, firefly luciferase, bacterialluciferase, β-galactosidase and alkaline phosphatase. In such an assay,the reporter gene is typically under control of a regulatory regione.g., a promoter, from the PFDN-4 gene.

The amount of expression is then compared to the amount of expression inthe absence of the test compound. A substantially identical cell may bederived from the same cells from which the recombinant cell was preparedbut which had not been modified by introduction of heterologous DNA. Adifference in the amount of expression indicates that the test compoundhas in some manner altered PFDN-4 levels.

In assays to identify PFDN-4 inhibitors, samples that are treated with apotential inhibitor are compared to control samples to determine theextent of modulation. Control samples (untreated with candidateinhibitors) are assigned a relative activity value of 100. Inhibition ofPFDN-4 is achieved when the activity value relative to the control isabout 80%, optionally 50%, optionally 25-0%.

Candidate Compounds

The compounds tested as inhibitors of PFDN-4 can be any small chemicalcompound, or a biological entity, e.g., a macromolecule such as aprotein, sugar, nucleic acid or lipid. Alternatively, modulators can begenetically altered versions of PFDN-4. Typically, test compounds willbe small chemical molecules and peptides or antibodies.

In some embodiments, the agents have a molecular weight of less than1,500 daltons, and in some cases less than 1,000, 800, 600, 500, or 400daltons. The relatively small size of the agents can be desirablebecause smaller molecules have a higher likelihood of havingphysiochemical properties compatible with good pharmacokineticcharacteristics, including oral absorption than agents with highermolecular weight. For example, agents less likely to be successful asdrugs based on permeability and solubility were described by Lipinski etal. as follows: having more than 5 H-bond donors (expressed as the sumof OHs and NHs); having a molecular weight over 500; having a LogP over5 (or MLogP over 4.15); and/or having more than 10 H-bond acceptors(expressed as the sum of Ns and Os). See, e.g., Lipinski et al. Adv DrugDelivery Res 23:3-25 (1997). Compound classes that are substrates forbiological transporters are typically exceptions to the rule.

Essentially any chemical compound can be used as a potential modulatoror ligand in the assays of the invention. Most often, compounds can bedissolved in aqueous or organic (especially DMSO-based) solutions. Theassays are designed to screen large chemical libraries by automating theassay steps, which are typically run in parallel (e.g., in microtiterformats on microtiter plates in robotic assays). It will be appreciatedthat there are many suppliers of chemical compounds, including Sigma(St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis,Mo.), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and thelike.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” are then screened inone or more assays, as described herein, to identify those librarymembers (particular chemical species or subclasses) that display adesired characteristic activity. The compounds thus identified can serveas conventional “lead compounds” or can themselves be used as potentialor actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat.Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagiharaet al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Russell & Sambrook, all supra),peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083),antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology,14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see,e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S. Pat. No.5,593,853), small organic molecule libraries (see, e.g.,benzodiazepines, Baum C&EN, January 18, page 33 (1993); isoprenoids,U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat.No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S.Pat. No. 5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, Mo., 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

Solid State and Soluble High Throughput Assays

In one embodiment the invention provides soluble assays using moleculessuch as a domain, e.g., a binding domain, a subunit association region,etc.; a domain that is covalently linked to a heterologous protein tocreate a chimeric molecule; a PFDN-4; or a cell or tissue expressing aPFDN-4, either naturally occurring or recombinant. In anotherembodiment, the invention provides solid phase based in vitro assays ina high throughput format, where the domain, chimeric molecule, PFDN-4,or cell or tissue expressing PFDN-4 is attached to a solid phasesubstrate. In high throughput screening assays, it is possible to screenup to several thousand different modulators or ligands in a single day.

The molecule of interest can be bound to the solid state component,directly or indirectly, via covalent or non covalent linkage e.g., via atag. The tag can be any of a variety of components. In general, amolecule that binds the tag (a tag binder) is fixed to a solid support,and the tagged molecule of interest (e.g., the prefoldin complex memberof interest) is attached to the solid support by interaction of the tagand the tag binder. A number of tags and tag binders can be used, basedupon known molecular interactions well described in the literature. Forexample, where a tag has a natural binder, for example, biotin, proteinA, or protein G, it can be used in conjunction with appropriate tagbinders (avidin, streptavidin, neutravidin, the Fc region of animmunoglobulin, etc.). Antibodies to molecules with natural binders suchas biotin are also widely available and are appropriate tag binders;see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis Mo.).Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Syntheticpolymers, such as polyurethanes, polyesters, polycarbonates, polyureas,polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes,polyimides, and polyacetates can also form an appropriate tag or tagbinder. Many other tag/tag binder pairs are also useful in assay systemsdescribed herein, as would be apparent to one of skill upon review ofthis disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly-gly sequencesof between about 5 and 200 amino acids. Such flexible linkers are knownto persons of skill in the art. For example, poly(ethelyne glycol)linkers are available from Shearwater Polymers, Inc. Huntsville, Ala.These linkers optionally have amide linkages, sulfhydryl linkages, orheterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature. See, e.g., Merrifield, J.Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)(describing synthesis of solid phase components on pins); Frank &Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719(1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

Computer-based Assays

Yet another assay for compounds that modulate PFDN-4 activity involvescomputer assisted drug design, in which a computer system is used togenerate a three-dimensional structure of PFDN-4 based on the structuralinformation encoded by the amino acid sequence. The input amino acidsequence interacts directly and actively with a pre-establishedalgorithm in a computer program to yield secondary, tertiary, andquaternary structural models of the protein. The models of the proteinstructure are then examined, for example, to identify the regions thathave the ability to bind other members of the prefoldin complex. Theseregions are then used to identify various compounds that inhibit PFDN-4activity.

The three-dimensional structural model of the protein is generated byentering protein amino acid sequences of at least 10 amino acid residuesor corresponding nucleic acid sequences encoding a PFDN-4 polypeptideinto the computer system. The amino acid sequence may comprise SEQ IDNO: 2. The amino acid sequence represents the primary sequence orsubsequence of the protein, which encodes the structural information ofthe protein. At least 10 residues of the amino acid sequence (or anucleotide sequence encoding 10 amino acids) are entered into thecomputer system from computer keyboards, computer readable substratesthat include, but are not limited to, electronic storage media (e.g.,magnetic diskettes, tapes, cartridges, and chips), optical media (e.g.,CD ROM), information distributed by internet sites, and by RAM. Thethree-dimensional structural model of the protein is then generated bythe interaction of the amino acid sequence and the computer system,using software known to those of skill in the art.

The software looks at certain parameters encoded by the primary sequenceto generate the structural model. These parameters are referred to as“energy terms,” and primarily include electrostatic potentials,hydrophobic potentials, solvent accessible surfaces, and hydrogenbonding. Secondary energy terms include van der Waals potentials.Biological molecules form the structures that minimize the energy termsin a cumulative fashion. The computer program is therefore using theseterms encoded by the primary structure or amino acid sequence to createthe secondary structural model.

The tertiary structure of the protein encoded by the secondary structureis then formed on the basis of the energy terms of the secondarystructure. The user at this point can enter additional variables such aswhether the protein is membrane bound or soluble and its cellularlocation, e.g., cytoplasmic. These variables along with the energy termsof the secondary structure are used to form the model of the tertiarystructure. In modeling the tertiary structure, the computer programmatches hydrophobic faces of secondary structure with like, andhydrophilic faces of secondary structure with like.

Once the structure has been generated, potential ligand binding regionsare identified by the computer system. Three-dimensional structures forpotential ligands are generated by entering amino acid or nucleotidesequences or chemical formulas of compounds, as described above. Thethree-dimensional structure of the potential ligand is then compared tothat of PFDN-4 to identify ligands that bind to the PFDN-4. Bindingaffinity between the protein and ligands is determined using energyterms to determine which ligands have an enhanced probability of bindingto the protein.

Expression Assays

Certain screening methods involve screening for a compound thatmodulates the expression of PFDN-4. Such methods generally involveconducting cell-based assays in which test compounds are contacted withone or more cells expressing a PFDN-4 and then detecting a decrease inexpression (either transcript or translation product). Such assays areoften performed with cells that overexpress PFDN-4.

Expression can be detected in a number of different ways. As describedherein, the expression levels of the protein in a cell can be determinedby probing the mRNA expressed in a cell with a probe that specificallyhybridizes with a PFDN-4 transcript (or complementary nucleic acidderived therefrom). Alternatively, protein can be detected usingimmunological methods in which a cell lysate is probed with antibodiesthat specifically bind to the protein.

Other cell-based assays are reporter assays conducted with cells that donot express the protein. Often, these assays are conducted with aheterologous nucleic acid construct that includes a promoter that isoperably linked to a reporter gene that encodes a detectable product. Anumber of different reporter genes can be utilized. Some reporters areinherently detectable. An example of such a reporter is greenfluorescent protein that emits fluorescence that can be detected with afluorescence detector. Other reporters generate a detectable product.Often such reporters are enzymes. Exemplary enzyme reporters include,but are not limited to, β-glucuronidase, CAT (chloramphenicol acetyltransferase), luciferase, β-galactosidase and alkaline phosphatase.

In these assays, cells harboring the reporter construct are contactedwith a test compound. A test compound that inhibits the activity of thepromoter, e.g., by binding to it or triggering a cascade that produces amolecule that decreases the promoter-induced expression of thedetectable reporter can be detected by comparison to control cells thathave not been treated with the inhibitor. Certain other reporter assaysare conducted with cells that harbor a heterologous construct thatincludes a transcriptional control element that activates expression ofPFDN-4 and a reporter operably linked thereto. Here, too, an agent thatbinds to the transcriptional control element to activate expression ofthe reporter or that triggers the formation of an agent that binds tothe transcriptional control element to activate reporter expression, canbe identified by the generation of signal associated with reporterexpression.

In another embodiment, PFDN-4are used to generate animal models ofcancer. For example, a transgenic animals can be generated thatoverexpresses PFDN-4. Depending on the desired expression level,promoters of various strengths can be employed to express the transgene.Also, the number of copies of the integrated transgene can be determinedand compared for a determination of the expression level of thetransgene. Animals generated by such methods can be used for screeningfor inhibitors to treat cancer. mo

Nucleic Acid Inhibitors

Screening assays of the invention often evaluate nucleic acid moleculesas potential inhibitors. For example, ribozymes, antisense RNA and/orsmall interfering RNA (siRNA) molecules can be screened for the abilityto decrease PFDN-4 levels.

I some embodiments, siRNA molecules designed to target PFDN-4 RNA arescreened. In mammalian cells, introduction of long dsRNA (>30 nt) ofteninitiates a potent antiviral response, exemplified by nonspecificinhibition of protein synthesis and RNA degradation. The phenomenon ofRNA interference is described and discussed, e.g., in Bass, Nature411:428-29 (2001); Elbahir et al., Nature 411:494-98 (2001); and Fire etal., Nature 391:806-11 (1998), where methods of making interfering RNAalso are discussed. The siRNAs based upon the PFDN-4 sequences disclosedherein are less than 100 base pairs, typically 30 bps or shorter, andare made by approaches known in the art. Exemplary siRNAs according tothe invention could have up to 29 bps, 25 bps, 22 bps, 21 bps, 20 bps,15 bps, 10 bps, 5 bps or any integer thereabout or therebetween.

The siRNA can comprise two complementary molecules, or can beconstructed such that a single transcript has both the sense andcomplementary antisense sequences from the target gene, e.g., a hairpin.

Methods for designing double stranded RNA to inhibit gene expression ina target cell are known (see, e.g., U.S. Pat. No. 6,506,559; Elbashir etal. Methods 26:199-213, 2002; Chalk et al., Biochem. Biophysy Res. Comm319:264-274, 2004; Cui et al. Computer Method and Programs inBiomedicine 75:67-73, 2004, Wang et al., Bioinformatics 20:1818-1820,2004). For example, design of siRNAs (including hairpins) typicallyfollow known thermodynamic rules (see, e.g., Schwarz, et al., Cell115:199-208, 2003; Reynolds et al., Nat Biotechnol. 22:326-30, 2004;Khvorova, et al., Cell 115:209-16, 2003). Many computer programs areavailable for selecting regions of PFDN-4 that are suitable targetsites. These include programs available through commercial sources suchas Ambion, Dharmacon, Promega, Invitrogen, Ziagen, and GenScript as wellas noncommercial sources such as EMBOSS, The Wistar Institute, WhiteheadInstitute, and others.

For example, design can be based on the following considerations.Typically shorter sequences, i.e., less than about 30 nucleotides areselected. The coding region of the mRNA is usually targeted. The searchfor an appropriate target sequence optionally begins 50-100 nucleotidesdownstream of the start codon, as untranslated region binding proteinsand/or translation initiation complexes may interfere with the bindingof the siRNP endonuclease complex. Some algorithms, e.g., based on thework of Elbashir et al., supra, search for a 23-nt sequence motifAA(N19)TT (N, any nucleotide) and select hits with approx. 50%G/C-content (30% to 70% has also worked in for them). If no suitablesequences are found, the search is extended using the motif NA(N21). Thesequence of the sense siRNA corresponds to (N19)TT or N21 (position 3 to23 of the 23-nt motif), respectively. In the latter case, the 3′ end ofthe sense siRNA is converted to TT.

Other algorithisms preferentially select siRNAs corresponding to thetarget motif NAR(N17)YNN, where R is purine (A, G) and Y is pyrimidine(C, U). The respective 21-nt sense and antisense siRNAs therefore beginwith a purine nucleotide and can also be expressed from pol IIIexpression vectors without a change in targeting site; expression ofRNAs from pol III promoters is only efficient when the first transcribednucleotide is a purine.

Other nucleic acids, e.g., ribozymes, antisense, can also be designedbased on known principles. For example, Sfold (see, e.g, Ding, et al.,Nucleic Acids Res. 32 Web Server issue, W135-W141, Ding & Lawrence,Nucl. Acids Res. 31: 7280, 7301, 2003; and Ding & Lawrence Nucl. AcidsRes. 20:1034-1046, 2001) provides programs relating to designingribozymes and antisense, as well as siRNAs.

Disease Treatment and Diagnosis/Prognosis

PFDN-4 nucleic acid and polypeptide sequences can be used for diagnosisor prognosis of cancer in a patient. For example, the sequence, level,or activity of PFDN-4 in a patient can be determined, wherein analteration, e.g., an increase in the level of expression or activity ofPFDN-4, or the detection of an increase in copy number or mutations inthe PFDN-4, indicates the presence or the likelihood of cancer.

Often, such methods will be used in conjunction with additionaldiagnostic methods, e.g., detection of other cancer indicators, e.g.,cell morphology, and the like. In other embodiments, a tissue sampleknown to contain cancerous cells, e.g., from a tumor, will be analyzedfor PFDN-4 levels to determine information about the cancer, e.g., theefficacy of certain treatments, the survival expectancy

In some embodiments, the level of PFDN-4 can be used to determine theprognosis of a patient with cancer. For example, if cancer is detectedusing a technique other than by detecting PFDN-4, e.g., tissue biopsy,then the presence or absence of PFDN-4 can be used to determine theprognosis for the patient, i.e., an elevated level of PFDN-4 willtypically indicate a reduced survival expectancy in the patient comparedto in a patient with cancer but with a normal level of PFDN-4. As usedherein, “survival expectancy” refers to a prediction regarding theseverity, duration, or progress of a disease, condition, or any symptomthereof. In a preferred embodiment, an increased level, a diagnosticpresence, or a quantified level, of PFDN-4 is statistically correlatedwith the observed progress of a disease, condition, or symptom in alarge number of patients, thereby providing a database wherefrom astatistically-based prognosis can be made. For example, in a particulartype of patient, a human of a particular age, gender, medical condition,medical history, etc., a detection of a level of PFDN-4 that is, e.g., 2fold higher than a control level may indicate, e.g., a 10% reducedsurvival expectancy in the human compared to in a similar human with anormal level of PFDN-4, based on a previous study of the level of PFDN-4in a large number of similar patients whose disease progression wasobserved and recorded.

The methods of the present invention can be used to determine theoptimal course of treatment in a patient with cancer. For example, thepresence of an elevated level of PFDN-4 can indicate a reduced survivalexpectancy of a patient with cancer, thereby indicating a moreaggressive treatment for the patient In addition, a correlation can bereadily established between levels of PFDN-4, or the presence or absenceof a diagnostic presence of PFDN-4, and the relative efficacy of one oranother anti-cancer agent. Such analyses can be performed, e.g.,retrospectively, i.e., by detecting PFDN-4 levels in samples takenpreviously from patients that have subsequently undergone one or moretypes of anti-cancer therapy, and correlating the PFDN-4 levels with theknown efficacy of the treatment.

Administration of Pharmaceutical and Vaccine Compositions

Inhibitors of PFDN-4 can be administered to a patient for the treatmentof cancer, e.g., ovarian cancer. As described in detail below, theinhibitors are administered in any suitable manner, optionally withpharmaceutically acceptable carriers.

The identified inhibitors can be administered to a patient attherapeutically effective doses to prevent, treat, or control cancer.The compounds are administered to a patient in an amount sufficient toelicit an effective protective or therapeutic response in the patient.An effective therapeutic response is a response that at least partiallyarrests or slows the symptoms or complications of the disease. An amountadequate to accomplish this is defined as “therapeutically effectivedose.” The dose will be determined by the efficacy of the particularPFDN-4 inhibitors employed and the condition of the subject, as well asthe body weight or surface area of the area to be treated. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse effects that accompany the administration of a particularcompound or vector in a particular subject.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, for example, by determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅₀ (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index and can be expressed as the ratio, LD₅₀/FD₅₀.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue to minimize potential damage to normal cellsand, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can beused to formulate a dosage range for use in humans. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage can varywithin this range depending upon the dosage form employed and the routeof administration. For any compound used in the methods of theinvention, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose can be formulated in animal models toachieve a circulating plasma concentration range that includes the IC₅₀(the concentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma can be measured, for example, by high performance liquidchromatography (HPLC). In general, the dose equivalent of a modulator isfrom about 1 ng/kg to 10 mg/kg for a typical subject.

Pharmaceutical compositions for use in the present invention can beformulated by standard techniques using one or more physiologicallyacceptable carriers or excipients. The compounds and theirphysiologically acceptable salts and solvates can be formulated foradministration by any suitable route, including via inhalation,topically, nasally, orally, parenterally (e.g., intravenously,intraperitoneally, intravesically or intrathecally) or rectally.

For oral administration, the pharmaceutical compositions can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients, including binding agents,for example, pregelatinised maize starch, polyvinylpyrrolidone, orhydroxypropyl methylcellulose; fillers, for example, lactose,microcrystalline cellulose, or calcium hydrogen phosphate; lubricants,for example, magnesium stearate, talc, or silica; disintegrants, forexample, potato starch or sodium starch glycolate; or wetting agents,for example, sodium lauryl sulphate. Tablets can be coated by methodswell known in the art. Liquid preparations for oral administration cantake the form of, for example, solutions, syrups, or suspensions, orthey can be presented as a dry product for constitution with water orother suitable vehicle before use. Such liquid preparations can beprepared by conventional means with pharmaceutically acceptableadditives, for example, suspending agents, for example, sorbitol syrup,cellulose derivatives, or hydrogenated edible fats; emulsifying agents,for example, lecithin or acacia; non-aqueous vehicles, for example,almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils;and preservatives, for example, methyl or propyl-p-hydroxybenzoates orsorbic acid. The preparations can also contain buffer salts, flavoring,coloring, and/or sweetening agents as appropriate. If desired,preparations for oral administration can be suitably formulated to givecontrolled release of the active compound.

For administration by inhalation, the compounds may be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, forexample, dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator can beformulated containing a powder mix of the compound and a suitable powderbase, for example, lactose or starch.

The compounds can be formulated for parenteral administration byinjection, for example, by bolus injection or continuous infusion.Formulations for injection can be presented in unit dosage form, forexample, in ampoules or in multi-dose containers, with an addedpreservative. The compositions can take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and can containformulatory agents, for example, suspending, stabilizing, and/ordispersing agents. Alternatively, the active ingredient can be in powderform for constitution with a suitable vehicle, for example, sterilepyrogen-free water, before use.

The compounds can also be formulated in rectal compositions, forexample, suppositories or retention enemas, for example, containingconventional suppository bases, for example, cocoa butter or otherglycerides.

Furthermore, the compounds can be formulated as a depot preparation.Such long-acting formulations can be administered by implantation (forexample, subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the compounds can be formulated withsuitable polymeric or hydrophobic materials (for example as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

The compositions can, if desired, be presented in a pack or dispenserdevice that can contain one or more unit dosage forms containing theactive ingredient. The pack can, for example, comprise metal or plasticfoil, for example, a blister pack. The pack or dispenser device can beaccompanied by instructions for administration.

Inhibitors of Gene Expression

In one aspect of the present invention, PFDN-4 inhibitors can alsocomprise nucleic acid molecules that inhibit expression of PFDN-4.Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding engineered PFDN-4 polypeptides inmammalian cells or target tissues, or alternatively, nucleic acids e.g.,inhibitors of PFDN-4 activity, such as siRNAs, ribozymes, or anti-senseRNAs. Non-viral vector delivery systems include DNA plasmids, nakednucleic acid, and nucleic acid complexed with a delivery vehicle such asa liposome. Viral vector delivery systems include DNA and RNA viruses,which have either episomal or integrated genomes after delivery to thecell. For a review of gene therapy procedures, see Anderson, Science256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani &Caskey, TIBTECH 11: 162-166 (1993); Dillon, TIBTECH 11: 167-175 (1993);Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiologyand Immunology Doerfler and Bohm (eds) (1995); and Yu et al., GeneTherapy 1: 13-26 (1994).

In some embodiments, siRNAs are administered. siRNA therapy is carriedout by administering to a patient a siRNA by standard vectors encodingthe siRNAs of the invention and/or gene delivery systems such as bydelivering the synthetic siRNA molecules. Typically, synthetic siRNAmolecules are chemically stabilized to prevent nuclease degradation invivo. Methods for preparing chemically stabilized RNA molecules are wellknown in the art. Typically, such molecules comprise modified backbonesand nucleotides to prevent the action of ribonucleases. Othermodifications are also possible, for example, cholesterol-conjugatedsiRNAs have shown improved pharmacological properties (see, e.g., Songet al. Nature Med. 9:347-351 (2003).

Non-viral Delivery Methods

Methods of non-viral delivery of nucleic acids encoding engineeredpolypeptides of the invention include lipofection, microinjection,biolistics, virosomes, liposomes, immunoliposomes, polycation orlipid:nucleic acid conjugates, naked DNA, artificial virions, andagent-enhanced uptake of DNA. Lipofection is described in e.g., U.S.Pat. No. 5,049,386, U.S. Pat. No. 4,946,787; and U.S. Pat. No.4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Felgner, WO 91/17424, WO 91/16024.Delivery can be to cells (ex vivo administration) or target tissues (invivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Viral Delivery Methods

The use of RNA or DNA viral based systems for the delivery of inhibitorsof PFDN-4 are known in the art. Conventional viral based systems for thedelivery of PFDN-4 nucleic acid inhibitors can include retroviral,lentivirus, adenoviral, adeno-associated and herpes simplex virusvectors for gene transfer.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type, e.g., a pancreas or breast tissue. A viral vector istypically modified to have specificity for a given cell type byexpressing a ligand as a fusion protein with a viral coat protein on theviruses outer surface. The ligand is chosen to have affinity for areceptor known to be present on the cell type of interest. For example,Han et al., PNAS 92:9747-9751 (1995), reported that Moloney murineleukemia virus can be modified to express human heregulin fused to gp70,and the recombinant virus infects certain human cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other pairs of virus expressing a ligand fusion protein and targetcell expressing a receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., Fab or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences thought to favor uptakeby specific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In some embodiments, cellsare isolated from the subject organism, transfected with PFDN-4inhibitor nucleic acids and re-infused back into the subject organism(e.g., patient). Various cell types suitable for ex vivo transfectionare well known to those of skill in the art (see, e.g., Freshney et al.,Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) andthe references cited therein for a discussion of how to isolate andculture cells from patients).

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic nucleic acids can also be administered directly to theorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered. Administration is by any of the routes normally usedfor introducing a molecule into ultimate contact with blood or tissuecells. Suitable methods of administering such nucleic acids areavailable and well known to those of skill in the art, and, althoughmore than one route can be used to administer a particular composition,a particular route can often provide a more immediate and more effectivereaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention, as described below (see, e.g., Remington'sPharmaceutical Sciences, 17th ed., 1989).

In some embodiments, PFDN-4 polypeptides and polynucleotides can also beadministered as vaccine compositions to stimulate an immune response,typically a cellular (CTL and/or HTL) response. Such vaccinecompositions can include, e.g., lipidated peptides (see, e.g.,Vitiello,A. et al., J. Clin. Invest. 95:341 (1995)), peptide compositionsencapsulated in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see,e.g., Eldridge, et al., Molec. Immunol. 28:287-294, (1991); Alonso etal., Vaccine 12:299-306 (1994); Jones et al., Vaccine 13:675-681(1995)), peptide compositions contained in immune stimulating complexes(ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875 (1990); Hu etal., Clin Exp Immunol. 113:235-243 (1998)), multiple antigen peptidesystems (MAPs) (see, e.g., Tam, Proc. Natl. Acad. Sci. U.S.A.85:5409-5413 (1988); Tam, J. Immunol. Methods 196:17-32 (1996)),peptides formulated as multivalent peptides; peptides for use inballistic delivery systems, typically crystallized peptides, viraldelivery vectors (Perkus, et al., In: Concepts in vaccine development(Kaufmann, ed., p. 379, 1996); Chakrabarti, et al., Nature 320:535(1986); Hu et al., Nature 320:537 (1986); Kieny, et al., AIDSBio/Technology 4:790 (1986); Top et al., J. Infect. Dis. 124:148 (1971);Chanda et al., Virology 175:535 (1990)), particles of viral or syntheticorigin (see, e.g., Kofler et al., J. Immunol. Methods. 192:25 (1996);Eldridge et al., Sem. Hematol. 30:16 (1993); Falo et al., Nature Med.7:649 (1995)), adjuvants (Warren et al., Annu. Rev. Immunol. 4:369(1986); Gupta et al., Vaccine 11:293 (1993)), liposomes (Reddy et al.,J. Immunol. 148:1585 (1992); Rock, Immunol. Today 17:131 (1996)), or,naked or particle absorbed cDNA (Ulmer, et al., Science 259:1745 (1993);Robinson et al., Vaccine 11:957 (1993); Shiver et al., In: Concepts invaccine development (Kaufmann, ed., p. 423, 1996); Cease & Berzofsky,Annu. Rev. Immunol. 12:923 (1994) and Eldridge et al., Sem. Hematol.30:16 (1993)). Toxin-targeted delivery technologies, also known asreceptor mediated targeting, such as those of Avant Immunotherapeutics,Inc. (Needham, Mass.) may also be used.

Kits for Use in Diagnostic and/or Prognostic Applications

For use in diagnostic, research, and therapeutic applications suggestedabove, kits are also provided by the invention. In the diagnostic andresearch applications such kits may include any or all of the following:assay reagents, buffers, cancer-specific nucleic acids or antibodies,hybridization probes and/or primers, antisense polynucleotides, siRNAs,ribozymes, dominant negative cancer polypeptides or polynucleotides,small molecules inhibitors of cancer-associated sequences etc. Atherapeutic product may include sterile saline or anotherpharmaceutically acceptable emulsion and suspension base.

In addition, the kits may include instructional materials containingdirections (i.e., protocols) for the practice of the methods of thisinvention. While the instructional materials typically comprise writtenor printed materials they are not limited to such. Any medium capable ofstoring such instructions and communicating them to an end user iscontemplated by this invention. Such media include, but are not limitedto electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. Such media mayinclude addresses to internet sites that provide such instructionalmaterials.

The present invention also provides for kits for screening formodulators of PFDN-4 cancer-associated sequences. Such kits can beprepared from readily available materials and reagents. For example,such kits can comprise one or more of the following materials: a PFDN-4cancer-associated polypeptide or polynucleotide, reaction tubes, andinstructions for testing PFDN-4 cancer-associated activity. Optionally,the kit contains biologically active PFDN-4 cancer protein. A widevariety of kits and components can be prepared according to the presentinvention, depending upon the intended user of the kit and theparticular needs of the user. Diagnosis would typically involveevaluation of a plurality of genes or products. The genes will beselected based on correlations with important parameters in diseasewhich may be identified in historical or outcome data.

EXAMPLES

Array Comparative Genome Hybridization (aCGH) was used to evaluatepancreatic islet cell tumors from RIP1-Tag2 transgenic mice. RIP1-Tag2mice express an oncogene, the SV40 large T antigen, that interferes withthe functions of the Rb and p53 tumor suppressors. This is necessary,but not sufficient to elicit tumors, in that tumorigenesis takes 12weeks and involves the sequential appearance of histological stagesinferred to involve genetic and epigenetic secondary events thatfacilitate tumor progression.

Array CGH data using RIP-Tag2 tumors identified a recurrent copy numbergain in chromosome 2, cytoband H3 with a frequency of 15-30% of tumors,depending on genetic background (Hager et al, 2003). This locus is ofparticular interest because it is syntenic to human 20q13.2, a regionfrequently amplified in a variety of human cancers, including pancreaticendocrine tumors (Stumpf 2000, Zhao 2001). In human cancers,amplifications of this locus are associated with a more aggressivephenotype, increased metastasis and poor clinical prognosis (Hidaka2000, Tanner 1995, Diebold 2000). Detailed genomic studies of humantumors have revealed a high degree of genomic complexity in thisamplicon (Collins 1998, Albertson 2000, Collins 2001).

The minimal region of amplification was defined by one tumor that had avery narrow gain of about 217 Kb, which contains BCAS1/NABC1, Cyp24,Prefoldin 4 (PFDN-4) and a putative pseudogene Ub16/SUMO. The broaderregion of recurrent gain encompasses the distal ˜20Mb of mousechromosome 2—and includes the mouse homolog of zinc-finger protein 217(Znf217), a candidate oncogene that maps within the amplicon identifiedin human tumors.

Expression of all of the candidate genes and putative pseudogenesmapping within the locus commonly gained/amplified in RIP-Tag mouse andhuman breast tumors was assessed. Expression was analyzed using pools oftumors and different cell lines derived from these tumors (PTC cells).It was found that the PFDN-4 gene was consistently upregulated (FIG.1A).

Of the genes in the amplified region, BCAS1, Cyp24 and 2 pseudogeneswere excluded, as they were not expressed in primary tumors or in anyislet tumor-derived PTC cell line (Table 1). Two genes were expressed inboth tumors and PTC lines: the mouse homolog of Znf217 and PFDN-4. Ofthese two candidates, Znf217 was expressed in some, but not all PTC celllines, and its expression levels did not correlate with the presence ofamplification of mouse chromosome: some non-CNG2 cell lines showedhigher expression levels than the CNG2-containing cell line PTC4 (notshown). On the other hand, PFDN-4 was expressed in all tumors and PTCcells tested, and its expression levels correlated with the presence ofCNG2 (in βTC4 its expression levels were 2-2.5 higher than in the otherβTC cell lines). TABLE 1 EVALUATION OF CANDIDATE GENES IN THE REGION OFCNG2 Expressed Within minimal in primary Expressed Gene CNG2? tumors? inβTC? Notes PFDN-4 Yes Yes All Increased expression correlates withpresence of CNG2 BCAS Yes No No Cyp24 Yes No No DOK5 ˜225 kb Yes AllControl gene outside distal of the CNG2 locus ZNF217 ˜200 kb Yes SomeCandidate proximal in human 20q13 amplicon

PFDN-4 is one of six component proteins that comprises the Prefoldinprechaperonin complex that contributes to the folding of certain newlysynthesized proteins, most notably actin and tubulin, major componentsof the cellular cytoskeleton. Quantitative RT-PCR analysis of the sixPrefoldin subunit genes in an islet tumor-derived cell line lackingmouse chromosome 2 aberrations showed PFDN-4 to be expressed at muchlower levels than the five prefoldin subunit genes (FIG. 1B), suggestingit might be limiting Prefoldin activity in normal islet P cells intumors that do not contain an amplification of the chromosome.

To further investigate PFDN-4 as the affected oncogene/tumor progressionfactor in this locus, a series of transfected cultured PTC cell lineslacking mouse chromosome 2 amplification that expressed distinctivelyelevated levels of PFDN-4 were developed. Two control cell lines werealso produced to complement the parental cell line. One control cellline carried an empty expression vector, while the other overexpressedDOK5, a gene on chromosome 2 that is 225 Kb distal to the region ofamplification. DOK5 was included as a control because it is amplified insome tumors with large-scale gains but is arguably not the candidateoncogene by virtue of its exclusion in the minimal region. TaqMan®analysis showed that the DOK5-transfectant and one of the PFDN-4transfectant clones (PFDN-4 clone3) overexpressed their respective gene˜50 fold compared to the basal parental and control cell lines; anotherPFDN-4-transfectant clone (PFDN-4 clone4) overexpressed to 10 foldlevels compared to the parental and control cell lines. We assayed thesecell lines both in vitro (FIG. 2) and via subcutaneous inoculation intoimmunodeficient Rag1-null mice to assess transplantation tumorigenicity(FIG. 3).

The cell culture studies with thee cell lines did not reveal anydifference in proliferation rates (scored as cells in S-phase pulselabeled with BrdU) as a consequence of overexpressing either PFDN-4 orDOK5 (not shown). However, the PFDN-4-expressing cells had a 3-5 folddecrease in apoptosis rates in culture, scored as cleaved-Caspase-3positively stained. DOK5 had not effect on either proliferation rate orfrequency of apoptosis. The cell lines were also analyzed for theability to grow in anchorage independent conditions in soft agar. Thehigh expressing PFDN-4 line produced more abundant and large colonies,which is consistent with a more highly transformed phenotype.

In addition to having a reduced frequency of apoptosis, PFDN-4over-expressing tumor cells also exhibiteds alterations in cell shapeand cytoskeleton in culture, assuming a morphology suggestive of anepithelial to mesenchymal transition, a hallmark of increased malignancyin progression of many carcinomas. Moreover, when assayed fortransplantation tumorigenicity, the PFDN-4-overexpressing cells showed asignificant increase in tumor ‘take’ and tumor growth rate when injectedsubcutaneously into Ragl-knockout mice, as compared to non-transfectedBTC lines, empty vector containing line, and the DOK5-overexpressingline, as summarized in Table 2 and FIG. 1, and from additional data notshown. As shown in FIG. 3, both PFDN-4-overexpressing clones formnedpalpable tumors with a shorter latency and evidenced a faster rate oftumor growth than control cells from the BTC line transfected with theempty vector. TABLE 2 Over- # cells inoc. Tumor take rate Cell Lineexpressing? s.c. into ear? (tumors/mice inj.) βTC-vector Empty 1 10⁶ 4/9vector,/n.a. βTC-PFDN4.4 10X-PFDN-4 1 10⁶ 12/12 βTC-PFDN4.3 50X-PFDN-4 110⁶ 8/9 βTC-DOK5 50X-DOK5 1 10⁶ 1/2

We also evaluated microarray profiling data on a series of human breastcancer cell lines that were genotyped for copy number at chromosome20q13 (FIG. 4). This analysis showed a correlation between increasedcopy number of the 20q13 locus and increased expression of PFDN-4.

As noted above, the CNG2 locus is syntenic to human 20q13 locus that isfrequently amplified in human cancers. To test the hypothesis thatPFDN-4 is also playing an important role in human tumors, we performedan ‘in silico’ analysis of PFDN-4 expression levels in apublicly-available human tumor expression arrays (Ramaswamy et al.,Proc. Natl. Acad. Sci USA 98:15149-15154). This dataset consists of atumor bank with normal control tissue from 14 different tissues oforigin ranging from epithelial tumors, mesenchimal tumors and lymphaticmalignancies. Consistent with the findings in RIP-Tag2 mouse model,expression levels of PFDN-4 were significantly higher in the tumorsamples compared to control samples (pooled all 218 tumor samples versuspooled all 90 normal tissues, p=0.001). Moreover, several tissues showedsignificant increases in PFDN-4 levels in the tumors. These included:breast adenocarcinoma (p=0.02), uterus adenoma (p=0.03), mesothelioma(p=0.02), and follicular lymphoma (p<0.001). In another dataset from thesame laboratory (Bhattacharjee, et al., Proc. Natl. Acad. Sci USA98:13790-13795, 2001), significant differences were found in 190 lungadenocarcinoma samples and 21 squamous carcinoma samples when comparedto 17 normal tissue samples (p=0.009 and p<0.001 respectively).

The data collectively implicate PFDN-4 as an important oncogene/tumorprogression factor underlying the recurrent copy number gains of theCNG2 locus and as playing a role in human malignancies.

The above examples are provided by way of illustration only and not byway of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Table of Exemplary Sequences SEQ ID NO:1 Human prefoldin 4polynucleotide sequence--cds 19-423 Accession number BC010953.1 1ggggagtcca gtcccaagat ggcggccacc atgaagaagg cggctgcaga agatgtcaat 61gttactttcg aagatcaaca aaagataaac aaatttgcac ggaatacaag tagaatcaca 121gagctgaagg aagaaataga agtaaaaaag aaacaactcc aaaacctaga agatgcttgt 181gatgacatca tgcttgcaga tgatgattgc ttaatgatac cttatcaaat tggtgatgtc 241ttcattagcc attctcaaga agaaacgcaa gaaatgttag aagaagcaaa gaaaaatttg 301caagaagaaa ttgacgcctt agaatccaga gtggaatcaa ttcagcgagt gttagcagat 361ttgaaagttc agttgtatgc aaaattcggg agcaacataa accttgaagc tgatgaaagt 421taaacatttt ataatacttt ttttatttgt ttaataaact tgaatattgt aaaaaaaaaa 481aaaaaaaaaa aaaaaaaaaa SEQ ID NO:2 Human prefoldin 4 polypeptidesequence; Accession number: Q9NQP4MAATMKKAAAEDVNVTFEDQQKINKFARNTSRITELKEEIEVKKKQLQNLEDACDDIMLADDDCLMIPYQIGDVFISHSQEETQEMLEEAKKNLQEEIDALESRVESIQRVLADLKVQLYAKFGSNINLEADES

1. A method of identifying a modulator of expression of acancer-assocaited polypeptide, the method comprising the steps of: (i)contacting a cell that expresses SEQ ID NO:2 with a candidate modulator;and (ii) determining the level of expression of SEQ ID NO:2.
 2. Themethod of claim 1, wherein the modulator is an siRNA.
 3. A method foridentifying a compound that modulates a cancer-associated polypeptide,the method comprising the steps of: (i) contacting the compound with apolypeptide of SEQ ID NO:2; and (ii) determining the functional effectof the compound upon the polypeptide.
 4. The method of claim 3, whereinthe compound is a small organic molecule.
 5. A method of inhibitingproliferation of a cancer cell that overexpresses a polypeptide havingthe amino acid sequence of SEQ ID NO:2, the method comprising the stepof contacting the cancer cell with a therapeutically effective amount ofan inhibitor of the polypeptide.
 6. The method of claim 4, wherein thecell has an amplification of 20q13.
 7. The method of claim 4, whereinthe inhibitor is a small organic molecule.
 8. The method of claim 4,wherein the inhibitor is an siRNA molecule.
 9. A method of detectingcancer in a biological sample from a patient, the method comprisingcontacting the sample with a polynucleotide that selectively hybridizesto a nucleic acid sequence encoding a polypeptide having the amino acidsequence of SEQ ID NO:2; and detecting an increase in the level of thenucleic acid sequence, relative to normal, thereby detecting thepresence of cancer in the patient.
 10. The method of claim 9, wherein inthe cancer comprises cells that have an amplification in the chromosomalregion 20q13.
 11. The method of claim 9, wherein the cancer is selectedfrom the group consisting of ovarian cancer, breast cancer, cervicalcancer, gastric adenocarcinomas, uroepithelial tumors, and islet tumors.12. The method of claim 9, wherein the detecting step comprisesdetecting an increase in copy number of the nucleic acid that encodesthe polypeptide.
 13. The method of claim 9, wherein the detecting stepcomprises detecting an mRNA that encodes the polypeptide.
 14. The methodof claim 9, wherein the patient is suspected of having cancer.
 15. Themethod of claim 9, wherein the patient is undergoing a therapeuticregimen to treat cancer.
 16. A method of detecting cancer in abiological sample from a patient, the method comprising detecting anincrease in the level of a polypeptide set forth in SEQ ID NO:2,relative to normal, thereby detecting the presence of cancer in thepatient.
 17. The method of claim 16, wherein the step of detecting anincrease in the level of the polypeptide comprises performing animmunoassay.
 18. A method of monitoring the efficacy of a therapeutictreatment of cancer, the method comprising the steps of: (i) providing abiological sample from a patient undergoing the therapeutic treatment;and (ii) detecting the level of: a polypeptide having an amino acidsequence of SEQ ID NO:2, or of a nucleic acid that encodes thepolypeptide, in the biological sample compared to a level in abiological sample from the patient prior to, or earlier in, thetherapeutic treatment, thereby monitoring the efficacy of the therapy.